Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins.

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Differential contributions of the proteasome, autophagy, and chaperones to the clearance of arsenite-induced protein aggregates in yeast.

The poisonous metalloid arsenite induces widespread misfolding and aggregation of nascent proteins in vivo, and this mode of toxic action might underlie its suspected role in the pathology of certain protein misfolding diseases. Evolutionarily conserved protein quality-control systems protect cells against arsenite-mediated proteotoxicity, and herein, we systematically assessed the contribution of the ubiquitin-proteasome system, the autophagy-vacuole pathway, and chaperone-mediated disaggregation to the clearance of arsenite-induced protein aggregates in Saccharomyces cerevisiae. We show that the ubiquitin-proteasome system is the main pathway that clears aggregates formed during arsenite stress and that cells depend on this pathway for optimal growth. The autophagy-vacuole pathway and chaperone-mediated disaggregation both contribute to clearance, but their roles appear less prominent than the ubiquitin-proteasome system. Our in vitro assays with purified components of the yeast disaggregating machinery demonstrated that chaperone binding to aggregates formed in the presence of arsenite is impaired. Hsp104 and Hsp70 chaperone activity was unaffected by arsenite, suggesting that this metalloid influences aggregate structure, making them less accessible for chaperone-mediated disaggregation. We further show that the defect in chaperone-mediated refolding of a model protein was abrogated in a cysteine-free version of the substrate, suggesting that arsenite directly modifies cysteines in non-native target proteins. In conclusion, our study sheds novel light on the differential contributions of protein quality-control systems to aggregate clearance and cell proliferation and extends our understanding of how these systems operate during arsenite stress.

Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones that bind to aggregation-prone polypeptides at stress conditions. sHsps encage these polypeptides in assemblies, shielding them from further aggregation. To facilitate their subsequent solubilization and refolding by Hsp70 (DnaK) and Hsp100 (ClpB) chaperones, first, sHsps need to dissociate from the assemblies. In most γ-proteobacteria, these functions are fulfilled by a single sHsp (IbpA), but in a subset of Enterobacterales, a two-protein sHsp (IbpA and IbpB) system has evolved. To gain insight into the emergence of complexity within this chaperone system, we reconstructed the phylogeny of γ-proteobacteria and their sHsps. We selected proteins representative of systems comprising either one or two sHsps and analysed their ability to form sHsps-substrate assemblies. All the tested IbpA proteins, but not IbpBs, stably interact with an aggregating substrate. Moreover, in Escherichia coli cells, ibpA but not ibpB suppress the growth defect associated with low DnaK level, which points to the major protective role of IbpA during the breakdown of protein quality control. We also examined how sHsps affect the association of Hsp70 with the assemblies at the initial phase of disaggregation and how they affect protein recovery after stress. Our results suggest that a single gene duplication event has given rise to the sHsp system consisting of a strong canonical binder, IbpA, and its non-canonical paralog IbpB that enhances sHsps dissociation from the assemblies. The cooperation between the sHsps reduces the demand for Hsp70 needed to outcompete them from the assemblies by promoting sHsps dissociation without compromising assembly formation at heat shock. This potentially increases the robustness and elasticity of sHsps protection against irreversible aggregation.

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding.

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.

Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.

Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization.

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.

Evolution towards simplicity in bacterial small heat shock protein system.

Evolution can tinker with multi-protein machines and replace them with simpler single-protein systems performing equivalent functions in an equally efficient manner. It is unclear how, on a molecular level, such simplification can arise. With ancestral reconstruction and biochemical analysis, we have traced the evolution of bacterial small heat shock proteins (sHsp), which help to refold proteins from aggregates using either two proteins with different functions (IbpA and IbpB) or a secondarily single sHsp that performs both functions in an equally efficient way. Secondarily single sHsp evolved from IbpA, an ancestor specialized in strong substrate binding. Evolution of an intermolecular binding site drove the alteration of substrate binding properties, as well as the formation of higher-order oligomers. Upon two mutations in the α-crystallin domain, secondarily single sHsp interacts with aggregated substrates less tightly. Paradoxically, less efficient binding positively influences the ability of sHsp to stimulate substrate refolding, since the dissociation of sHps from aggregates is required to initiate Hsp70-Hsp100-dependent substrate refolding. After the loss of a partner, IbpA took over its role in facilitating the sHsp dissociation from an aggregate by weakening the interaction with the substrate, which became beneficial for the refolding process. We show that the same two amino acids introduced in modern-day systems define whether the IbpA acts as a single sHsp or obligatorily cooperates with an IbpB partner. Our discoveries illuminate how one sequence has evolved to encode functions previously performed by two distinct proteins.

The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.

The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

Chaperones in control of protein disaggregation.

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.

The Small Ones Matter-sHsps in the Bacterial Chaperone Network.

Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress. sHsps are defined not only by their relatively low molecular weight, but also by the presence of a conserved α-crystallin domain, which is flanked by less conserved, mostly unstructured, N- and C-terminal domains. sHsps form oligomers of different sizes which deoligomerize upon stress conditions into smaller active forms. Activated sHsps bind to aggregation-prone protein substrates to form assemblies that keep substrates from irreversible aggregation. Formation of these assemblies facilitates subsequent Hsp70 and Hsp100 chaperone-dependent disaggregation and substrate refolding into native species. This mini review discusses what is known about the role and place of bacterial sHsps in the chaperone network.

Two Bacterial Small Heat Shock Proteins, IbpA and IbpB, Form a Functional Heterodimer.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. ß-hairpins formed by strands ß5 and ß7 of IbpA or IbpB crystallin domains associate with the other one's ß-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.

Small but mighty: a functional look at bacterial sHSPs.

Small heat shock proteins (sHSPs) are widespread in every kingdom of life, being indispensable for protein quality control networks. Alongside canonical chaperone functions, sHSPs seem to have been a very plastic scaffold for acquiring multiple related functions across evolution. This review aims to summarize what is known about sHSPs functioning in the Bacteria Kingdom.

Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase.

Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell. In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.

Small heat shock proteins: multifaceted proteins with important implications for life.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

Function, evolution, and structure of J-domain proteins.

Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.

Conformational properties of aggregated polypeptides determine ClpB-dependence in the disaggregation process.

Severe thermal stress induces massive intracellular protein aggregation. The concerted action of Hsp70 (DnaK, DnaJ, GrpE) and Hsp100 (ClpB) chaperones results in solubilization of aggregates followed by reactivation of proteins. It was shown that the Hsp70 chaperone system works at the initial step of the disaggregation reaction and is able to disentangle polypeptides from aggregates. Studies of the protein disaggregation reaction performed in vitro showed that ClpB may be dispensable in disaggregation of certain proteins and/or aggregates of certain size. Here we focus our attention on those properties of firefly luciferase aggregates, which determine whether ClpB chaperone is required in the disaggregation process. We report that the size of the aggregates is not a major determinant. Instead, we postulate that certain conformational properties (in particular, beta-structures) of subunits forming these aggregates are the most important factor determining the necessity of the ClpB chaperone in the disaggregation process.

Hsp78 chaperone functions in restoration of mitochondrial network following heat stress.

Under physiological conditions mitochondria of yeast Saccharomyces cerevisiae form a branched tubular network, the continuity of which is maintained by balanced membrane fusion and fission processes. Here, we show using mitochondrial matrix targeted green fluorescent protein that exposure of cells to extreme heat shock led to dramatic changes in mitochondrial morphology, as tubular network disintegrated into several fragmented vesicles. Interestingly, this fragmentation did not affect mitochondrial ability to maintain the membrane potential. Cells subjected to recovery at physiological temperature were able to restore the mitochondrial network, as long as an active matrix chaperone, Hsp78, was present. Deletion of HSP78 gene did not affect fragmentation of mitochondria upon heat stress, but significantly inhibited ability to restore mitochondrial network. Changes of mitochondrial morphology correlated with aggregation of mitochondrial proteins. On the other hand, recovery of mitochondrial network correlated with disappearance of protein aggregates and reactivation of enzymatic activity of a model thermo-sensitive protein: mitochondrial DNA polymerase. Since protein disaggregation and refolding is mediated by Hsp78 chaperone collaborating with Hsp70 chaperone system, we postulate that effect of Hsp78 on mitochondrial morphology upon recovery after heat shock is mediated by its ability to restore activity of unknown protein(s) responsible for maintenance of mitochondrial morphology.

Expression of heat shock protein 72 in peritoneal leukocytes is induced by peritoneal dialysis.

BACKGROUND: One of the main limitations of peritoneal dialysis (PD) is deterioration of functional and morphological characteristics of the peritoneum. This complication appears to be related to the low biocompatibility profile of PD fluids. Recently, induction of the heat shock protein (HSP) stress response was demonstrated in cultured human mesothelial cells exposed to PD fluid in vitro. We investigated whether expression of heat shock protein 72 (HSP-72) in peritoneal macrophages is induced upon exposure to PD fluid during continuous ambulatory PD. METHODS: Peritoneal leukocytes were isolated from 4-hour dwell dialysate; peripheral blood mononuclear cells (PBMC) and peripheral blood monocytes isolated from the same patients were used as a control. In separate experiments, PBMC from healthy individuals were exposed in vitro to different PD fluids or to culture media. Expression of HSP-72 was assessed by Western immunoblotting, flow cytometry, and reverse-transcription polymerase chain reaction analysis. RESULTS: Macrophages and leukocytes isolated from dialysis effluent expressed significantly increased HSP-72 and mRNA levels compared to blood monocytes and PBMC of the same patients. In vitro exposure of PBMC to fresh PD fluids resulted in significantly higher expression of HSP-72 compared to those incubated in culture medium. PBMC exposed in vitro to standard lactate-buffered dialysis fluids also expressed significantly more HSP-72 compared to cells exposed to bicarbonate/lactate-buffered fluids. CONCLUSION: Our results indicate that exposure to PD fluids during dialysis triggers a shock response in peritoneal cells, which is manifested by significantly increased HSP-72 expression at both protein and mRNA levels. Analysis of this protein expression in peritoneal macrophages could be a new, convenient, and relevant way to assess the biocompatibility of PD fluids ex vivo.